Adipocyte preparation and extraction procedures have been developed to allow demonstration of important mechanisms of inhibition of lipolysis by adenosine. An improved assay for adipocyte cyclic AMP dependent protein kinase (A-kinase) has been developed, which allows for accurate measurement of the cellular state of activation of A-kinase. Acute removal of adenosine (using adenosine deaminase) from adipocyte preparations is found to lead to rapid, transient activation of A-kinase. In the quiescent, resting state (inhibited by nanomolar concentrations of adenosine) adipocytes show a very low fractional activation of A-kinase, much lower than reported heretofore. After the activation of adipocyte A-kinase, by removal of adenosine, has relaxed to resting levels, lipolysis still continues at linear rates and may be inhibited by the addition of the adenosine-analog N6-phenyliso-propyladenosine in an apparently cyclic AMP- and A-kinase-independent manner.